SUMMARY OF WORK Cardiac cell loss marks the transition from hypertrophy to heart failure and is the likely result of chronic myocardial ischemia and cell hypoxia. Cell loss is due predominantly to the death of cardiac myocytes and is mediated in part by apoptosis. Because adult cardiac myocytes are terminally differentiated cells, the effects of such loss can never be fully compensated. The identification of the intracellular signaling events and extracellular factors that regulate this process and the development of strategies to prevent such loss is, therefore, likely to have important beneficial consequences. We have adopted an experimental system to induce cardiomyocyte cell death by apoptosis that involves exposing neonatal cardiomyocytes to prolonged hypoxia. We show that in these cells there is increased expression and transactivating ability by the tumor suppressor gene, p53, that accompanies the onset of apoptotic cell death. In addition, expression of p21/WAF1, a downstream target of p53, also increases during hypoxia. Forced expression of either p53 or p21/WAF1 in normoxic myocytes through infection with recombinant adenoviruses leads to apoptosis, as does the incubation of normoxic myocytes with bafilomycin, an inhibitor of membrane-associated proton pumps which, in other cell types, leads to intracellular acidification. Apoptosis induced by p53, p21, and bafilomycin is effectively prevented or delayed by exposure to the hypertrophy-inducing factor, phenylephrine (PE). The PE rescue of apotosis occurs through a PI3-K-dependent pathway. The demonstration that p53 and p21 are both involved in the apoptosis of cardiac myocytes was unexpected since these cells are already growth arrested. Despite this, hypoxia, p53- or p21-infected, and bafilomycin-treated myocytes express high levels of c-myc mRNA and E2F DNA binding activity. Our current studies are directed at understanding how p53/p21 engage the "death machinery" in cardiomyocytes and in developing viral vectors to counteract the effects of p53.